Evaluating the sampling effort for the metabarcoding‐based detection of fish environmental DNA in the open ocean

Abstract Clarifying the effect of the sampling protocol on the detection of environmental DNA (eDNA) is essential for appropriately designing biodiversity research. However, technical issues influencing eDNA detection in the open ocean, which consists of water masses with varying environmental conditions, have not been thoroughly investigated. This study evaluated the sampling effort for the metabarcoding‐based detection of fish eDNA using replicate sampling with filters of different pore sizes (0.22 and 0.45 μm) in the subtropical and subarctic northwestern Pacific Ocean and Arctic Chukchi Sea. The asymptotic analysis predicted that the accumulation curves for detected taxa did not saturate in most cases, indicating that our sampling effort (7 or 8 replicates, corresponding to 10.5–40 L of filtration in total) was insufficient to fully assess the species diversity in the open ocean and that tens of replicates or a substantial filtration volume were required. The Jaccard dissimilarities between filtration replicates were comparable with those between the filter types at any site. In subtropical and subarctic sites, turnover dominated the dissimilarity, suggesting that the filter pore size had a negligible effect. In contrast, nestedness dominated the dissimilarity in the Chukchi Sea, implying that the 0.22 μm filter could collect a broader range of eDNA than the 0.45 μm filter. Therefore, the effect of filter selection on the collection of fish eDNA likely varies depending on the region. These findings highlight the highly stochastic nature of fish eDNA collection in the open ocean and the difficulty of standardizing the sampling protocol across various water masses.

partially shared candidate species and showed high sequence similarity in some pairs (over 98.5%), there was a risk of artificially inflating the taxonomic diversity when each resultant OTU was regarded as a distinct species. However, considering that Exocoetidae lack sufficient interspecific variation in the 12S ribosomal RNA gene region of mitochondrial DNA to distinguish species, merging these OTUs would result in an underestimation of the true diversity of Exocoetidae. Given the discrepancies between genetically distinct OTUs and the known species recognized by traditional taxonomy, evaluating the true diversity of the Exocoetidae from their eDNA is extremely difficult.
In this study, the OTUs assigned to Exocoetidae sp. 1-8 were regarded as distinct species, prioritizing the possibility that they originated from different species. Exocoetidae are pelagic fishes with a wide range of distribution in the open ocean. Because 28 exocoetids have been reported in one of our study areas, the waters surrounding Japan (Motomura, 2020), eDNA from multiple exocoetids is expected to be detected sympatrically. Furthermore, the difficulty of species-level discrimination in Exocoetidae is most likely due to the lack of interspecific variation rather than a high degree of intraspecific variation.
In this situation, taxonomic assignment based on sequence similarity is more likely to lead to merging errors than splitting errors. Therefore, this decision implies that the risk of splitting error due to insufficient interspecific variation was accepted rather than lowering the sensitivity to detect species due to merging error. Figure S1 Accumulation curves of the number of detected taxa against the number of sequencing reads across the four study sites (P03, K2,St.35,and St.10) and two different types of filters (022_Sterivex: 0.22 µm Sterivex-GV and 045_Sterivex: 0.45 µm Sterivex-HV). Read counts were corrected proportionally to the volume of PCR product mixed in a sequencing sample. Because the data was obtained across three sequencing runs, this correction was individually applied to each run. The symbols indicate the observed value, and the solid and dotted lines indicate the rarefaction and extrapolation curves, respectively.  Rarefaction (solid line segment) and extrapolation (broken line segment) curves with 95% confidence intervals (shaded areas) for taxonomic richness derived from data on fish taxonomic composition detected by eDNA metabarcoding with respect to the number of filtration replicates. The color of the line, area, and symbol corresponds to the filter types: 0.22 µm Sterivex-GV (022_Sterivex) and 0.45 µm Sterivex-HV (045_Sterivex) were denoted by red and blue, respectively. The symbols on the lines represent the observed value after aggregating all filtration replicates. Vertical dotted lines below the curves indicate the smallest accumulated filtration volume required to cover ≥95% of the Chao 2 estimator. Box plots showing (a) maximum filtration volume per filter (L) and (b) mean filtration speed (mL/min) when using the 0.45 µm Sterivex-HV filter (045_Sterivex) unit for vacuum filtration of seawater collected in the open ocean. NP, BS, and AO denote the Northwestern Pacific Ocean, the Bering Sea, and the Arctic Ocean, respectively. The bold lines in the boxes indicate medians, and the hinges of the boxes indicate the interquartile range (IQR; the first and third quartiles). Whiskers extend to the largest or smallest values no further than 1.5 ×IQR from the hinge. Black dots represent outliers with values greater than 1.5 ×IQR. Table S1 The number of sequencing reads remained after each step of bioinformatic processing. Detailed information about the procedure used in this study is provided in the manuscript. 022_Srteivex and 045_Sterivex denote 0.22 µm Sterivex-GV and 0.45 µm Sterivex-HV, respectively. The result of P03-045-1 was excluded from the analysis due to an unintended filtration volume. NA indicates not applicable.

Table S2
Summary of taxonomic assignment results obtained by the QCauto algorithm (Tanabe and Toju 2013) and modifications based on distribution knowledge. Candidate species names are consistent with scientific names adopted in the NCBI taxonomy database.

Table S8
Results of permutational dispersion test (PERMDISP) based on the Jaccard dissimilarity to test multivariate homogeneity of group dispersions in fish taxonomic composition obtained by eDNA metabarcoding grouped by filtration replicates.